The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Cells with FA deficiency are hypersensitive to DNA damage agents such as cisplatin and mitomycin C (MMC). BRCA genes collaborate with other genes in the same repair pathway, the FA pathway, to form foci of DNA repair on chromatin following DNA damage induction, or during the S phase of cell cycle. Given the large number of genes involved in this pathway, we hypothesize that a substantial proportion of patients with lung cancer harbor somatic genetic or epigenetic alterations resulting in defective FA homologous recombination repair. These patients are more likely to derive benefit from interstrand DNA crosslink agents. A test that could examine the functionality of this pathway as a whole and that could be practically applied for large-scale screening would be necessary to identify these patients. We have recently reported a FA triple-staining immunofluorescence (FATSI) test to evaluate the presence or absence of FANCD2 foci, and have generated preliminary data for somatic deficiency of this pathway in patients' tumors across several organ sites that include lung cancers. However, the specific genetic and epigenetic alterations that lead to inactivation of FA pathway in these sporadic tumors are still unknown. In order to provide sufficient molecular evidence to support such selection for clinical treatment and to understand the genetic and epigenetic changes that lead to FA pathway deficiency in lung cancers, we propose to accomplish following aims: (1) to identify FANCD2 foci defective tumor in lung cancer patients by analyzing 100 human lung tumor and 100 matching non-tumor samples, and (2) to perform a functional genomics analysis of FANCD2 foci defective tumors identified in aim 1 through analysis of the RNA transcriptome associated with the FA pathway in the tumor and adjacent non-tumor tissues, using next generation sequencing (RNAseq), and (3) to investigate promoter methylation of FA genes in FANCD2 foci defective tumors identified in aim 1. The information obtained from this project is expected to be useful for validating the immunofluorescence based triple staining test which can be used for justifying subsequent clinical therapeutic treatment for the patient with lung cancer.